Oocytes of Xenopus synthesize a specific type of 5S RNA, which is not made in somatic cells. The DNA containing the genes for this RNA (5S DNA) has been isolated from X. laevis and X. mulleri. The proposed program is to take advantage of what is known about the structure of these genes in a study of the mechanisms by which they are regulated in development. The first phase involves completion of a current study of the organization of X. mulleri 5S DNA. The repeating units of this DNA will be characterized by analysis using electron microscopy and gel electrophoresis, of the products of digestion of the purified 5S DNA by restriction endonucleases. Single chromosomal loci will be investigated in more detail by incorporation of restriction fragments, carrying several repeats, into bacterial plasmids, followed by cloning and amplification of individual fragments for analysis. The second and more extensive phase consists of a study of the chromatin containing the 5S genes, and its relationship to the regulation of 5S RNA synthesis. Information on the chromatin structure will be sought by determining the susceptibility of 5S DNA in chromatin to staphylococcal nuclease, and to restriction-enzymes having known modes of attack on deproteinized 5S DNA. Chromatin from cells in which oocyte-type 5S genes are active will be compared with those in which it is not. If the action of restriction enzymes on DNA in chromatin approximates that on free DNA, this property and a chromatin fractionation technique based on DNA molecular weight will be combined in a procedure for purifying 5S chromatin. Its chemical and template properties will be investigated. We expect the chromatin experiments to demonstrate conclusively the presence of 5S DNA in chromatin subunits from blood and liver. We shall investigate the same phenomenon in developing oocytes, in which these genes are active. We also expect to make good progress on experiments examining the detailed enzyme digestion of chromatin and by use of differential hybridization techniques. BIBLIOGRAPHIC REFERENCES: Carroll, D. and Brown, D.D. (1976). Cell, 7, 467-475. "Repeating Units of Xenopus laevis Oocyte-type 5S DNA are Heterogeneous in length."